FIG 2.

Generation of in-frame ΔpcrSR deletion and pcrSR-complemented mutants. (A) Scale model of genes coding for the two-component system PcrSR and parts of the 3′- and 5′-neighboring regions on the chromosome of A. aromaticum EbN1, displaying the wild type (top) and the ΔpcrSR mutant (bottom). Chromosomal hybridization locations of primer pairs used for construction of the knockout vector pk19 Ωhcl2/3ʹ-IR-pcrR are in gray, while those for the complementation plasmid pBBR1MCS-2 ΩpcrSR are indicated in black. The hybridization positions of the gene-specific primer pairs for pcrS and pcrR, as well as the primer pair ΔpcrSR_F/_R for identifying the knockout genotype, are in brown (see Table S1 in the supplemental material). Expected lengths of the respective PCR products are shown below the model. (B) Electropherogram of PCR products obtained from the wild type, the ΔpcrSR mutant, and the pcrSR-complemented (pcrSR compl.) mutant using specifically designed primer pairs. Applying the primer pair ΔpcrSR_F/_R, a 3,838-bp amplicon was obtained from the chromosome of the wild type, whereas the ΔpcrSR mutant and the pcrSR-complemented mutant yielded shorter, 310-bp amplicons. Correspondingly, amplification of pcrS and pcrR was possible only from the wild type and the pcrSR-complemented mutant but not from the ΔpcrSR mutant.